Glycerolipidic compounds used for the transfer of an active substance into a target cell

ABSTRACT

The invention concerns novel of compounds of formula (I) in which: R 1  and R 2 , identical or different are C 6 -C 23  alkyl or alkenyl radicals, linear or branched or —C(═O)-(C 6 -C 23 ) alkyl or —C(═O)-(C 6 -C 23 ) alkenyl, linear or branched, X is the oxygen atom or an amino —NR 3  radical, R 3  being a hydrogen atom or an inferior alkyl radical of 1 to 4 carbon atoms; n is a positive whole number from 1 to 6; m is a positive whole number from 1 to 6, and when n&gt;1, m can be identical or different. The invention also concerns novel compositions of said compounds and of active substances in particular therapeutically, comprising at least a negative charge for inserting said active substances in cells. It concerns in particular novel complexes, of which the active substance consists of one or several nucleic acids, useful for cell transfection.

The present invention relates to new glycero-lipid compounds and newcompositions containing them. More particularly, the present inventionrelates to the use of said compounds or of said compositions to preparea vector for transferring an active substance, in particular atherapeutically active substance comprising negative charges, inparticular a polynucleotide, into a target cell, particularly avertebrate cell, and more particularly a mammalian cell.

The transfer of a gene into a given cell is the very basis of genetherapy. This new technology, whose field of application is vast, makesit possible to envisage the treatment of serious diseases for which theconventional therapeutic alternatives are not very effective, or areeven inexistent, and applies to diseases which are either of geneticorigin (hemophilia, cystic fibrosis, myopathy and the like) or acquired(cancer, AIDS and the like).

During the past 30 years, numerous tools have been developed which allowthe introduction of various heterologous genes into cells, in particularmammalian cells. These different techniques may be divided into twocategories. The first category relates to physical techniques such asmicroinjection, electroporation or particle bombardment which, althougheffective, are greatly limited to applications in vitro and whoseimplementation is cumbersome and delicate. The second category involvestechniques relating to molecular and cell biology in which the gene tobe transferred is combined with a vector of a biological or syntheticnature which promotes the introduction of said material.

Currently, the most effective vectors are viral, in particularadenoviral or retroviral, vectors. The techniques developed are based onthe natural properties which these viruses have to cross the cellmembranes, to escape degradation of their genetic material and to causetheir genome to penetrate into the nucleus. These viruses have alreadybeen the subject of numerous studies and some of them are already usedexperimentally as vectors for genes in humans for the purpose, forexample, of a vaccination, an immunotherapy or a therapy intended tomake up for a genetic deficiency. However, this viral approach has manylimitations, in particular because of the limited capacity for cloninginto the viral genome, the risks of spreading in the host organism andin the environment the infectious viral particles produced, the risk ofartefactual mutagenesis by insertion into the host cell in the case ofretroviral vectors, and the high induction of immune and inflammatoryresponses in vivo during the therapeutic treatment, considerablylimiting the number of administrations which can be envisaged (McCoy etal., 1995, Human Gene Therapy, 6, 1553-1560; Yang et al., 1996,Immunity, 1, 433-442). These numerous disadvantages, in particular inthe context of a use in humans, have led several teams to developalternative systems of transferring polynucleotides.

Several non-viral methods are currently available. By way of example,there may be mentioned coprecipitation with calcium phosphate, the useof receptors mimicking viral systems (for a review see Cotten andWagner, 1993, Current Opinion in Biotechnology, 4, 705-710), or the useof polymers such as polyamidoamine (Haensler and Szoka, 1993,Bioconjugate Chem., 4, 372-379) or of polymer such as those presented inWO 95/24221 describing the use of dendritic polymers, the document WO96/02655 describing the use of polyethyleneimine, or ofpolypropyleneimine and the documents U.S. Pat. No. 5,595,897 and FR2,719,316 describing the use of conjugates of polylysine. Othernon-viral techniques are based on the use of liposomes whose value asagent allowing the introduction, into cells, of certain biologicalmacromolecules, such as for example DNA, RNA, proteins or certainpharmaceutically active substances, has been widely described in theliterature. To this end, several teams have already proposed the use ofcationic lipids which have a high affinity for the cell membranes and/orthe nucleic acids. Indeed, although it has been shown, in the case ofnucleic acids, that this type of macromolecule is capable of crossingthe plasma membrane of some cells in vivo (WO 90/11092), it isnevertheless the case that the observed transfection efficiency is stillhighly limited, because of in particular the polyanionic nature of thenucleic acids which prevent their passage across the cell membrane,which itself has a negative net apparent charge. Since 1989 (Felgner etal., Nature, 337, 387-388), cationic lipids have been presented asmolecules which are advantageous for promoting the introduction of largeanionic molecules, such as nucleic acids, into certain cells. Thesecationic lipids are capable of complexing anionic molecules, thustending to neutralize the negative charges on said molecules and topromote their coming close to the cells. Many teams have alreadydeveloped various cationic lipids. By way of example, there may bementioned DOTMA (Felgner et al., 1987, PNAS, 84, 7413-7417), DOGS orTransfectam™ (Behr et al., 1989, PNAS, 86, 6982-6986), DMRIE and DORIE(Felgner et al., 1993, Methods 5, 67-75), DC-CHOL (Gao et Huang, 1991,BBRC, 179, 280-285), DOTAP™ (McLachlan et al., 1995, Gene Therapy,2,674-622) or Lipofectamine™, as well as those described in PatentApplications WO9116024 or WO9514651.

More particularly, Application WO9405624 describes cationic lipids offormula:

in which the R radicals are in particular octadecenyl radicals, the Zradicals are C₁-C₁₃ alkyl or —C(═O)-(C₁-C₁₃) alkyl or acyl radicals, qis an integer from 1 to 6, X is in particular a short polyamine chain,such as spermine, spermidine, carboxyspermine or polylysine.

Applications EP 685457 and EP 685234 describe in particular cationiccompounds of formula:

in which R is in particular a hydrocarbon chain having 10 to 30 carbonatoms, saturated or otherwise, A may in particular be chosen from thegroups —O—(C═O)— and —NH—(C═O)—, n varies from 0 to 4 and R₁ is an alkylor a short aminoalkyl chain in which the primary amine is substitutedwith an alkyl having 2 to 8 carbon atoms. These compounds have a lowhemolytic activity and make it possible in vitro to introduce intoHelaS3 cells a double-stranded RNA capable of acting as growthinhibiting agent.

Application DE 19521412 describes compounds which also comprise anonpolar part and a polar part of formula:

in which p varies from 1 to 6, q varies from 0 to 2, R is eitherC(═O)-C1-23 or C1-23, saturated or otherwise, and Z is a peptide, anaminoacid or a branched amino structure. These cationic compounds allowthe in vitro transfection of cells in culture.

However, several studies (by way of example, see Mahato et al., J.Pharm. Sci., 1995, 84, 1267-1271, Thierry et al., 1995, P.N.A.S., 92,9742-9746) have demonstrated that the efficiency of transferring theanionic macromolecule into cells could vary depending in particular onthe interaction between the complexes and the cell membranes, the cellconsidered, the lipid composition of the cationic compounds, the size ofthe complexes formed with the anionic molecules and more particularlythe ratio between the positive and negative charges on the differentcomponents of said complex. The mechanisms which allow in particular theinteraction of the complexes with the cell membranes and the transfer ofthe complexes into the cell are still to a large extent poorlyunderstood and researchers proceed in their studies based on a highlyempirical approach. It is consequently desirable to provide othercationic lipids possibly having improved properties or properties whichare different from the cationic lipids already described.

The Applicant has now identified new glycerolipid compounds, which canbe provided in cationic form, useful in particular for transferring anactive substance comprising negative charges, in particular apolynucleotide, into a target cell, whose use may be envisaged inparticular in vivo in the context of a gene therapy.

Accordingly, the subject of the present invention is first of all acompound of formula:

in which:

R₁ and R₂, which are identical or different, are alkyl or alkenylradicals having 6 to 23 carbon atoms (noted C₆-C₂₃), which are linear orbranched, or radicals —C(═O)-(C₆-C₂₃) alkyl or —C(═O)-(C₆-C₂₃) alkenyl,or more particularly —C(═O)-(C₁₂-C₂₀) alkyl or —C(═O)-(C₁₂-C₂₀) alkenyl,which are linear or branched, aryl radicals, cycloalkyl radicals,fluoroalkyl radicals, oxyethylene or oxymethylene groups which areoptionally repeated, linear or branched, optionally substituted,

X is an oxygen atom or an amino radical —NR₃, R₃ being a hydrogen atomor an alkyl radical having 1 to 4 carbon atoms,

n is a positive integer from 1 to 6, preferably from 2 to 4,

m is a positive integer from 1 to 6, preferably from 2 to 4, and whenn>1, m may be identical or different from said n.

The term “alkenyl” is intended to indicate that the carbon chain inquestion may comprise one or more double bond(s) along said chain.

According to a specific case of the invention, said compounds arecharacterized in that R1 and/or R2 are fluorinated, that is to say thatat least one carbon of the polycarbon chain is substituted by afluorinated group. Examples of such molecules are provided in Example N.In this specific case, the number of fluorinated carbon atoms on eachchain R₁ or R₂ may vary from 1 to 12, and more particularly from 4 to 8,and is preferably 4. According to an advantageous case, R1 and/or R2 arealkyl radicals having 15 carbon atoms and the number of fluorinatedcarbon atoms is 4 for each of the relevant chains R₁ and R₂. The numberof fluorinated groups present on the chains R₁ and/or R₂ may inparticular vary from 1 to 23, more particularly from 9 to 17 andpreferably is 9.

The compounds according to the invention may, in addition, besubstituted. Such substitutions may in particular consist of a labelingmolecule (see labeling molecules in U.S. Pat. No. 4,711,955) which makesit possible, for example, to visualize the distribution of the compoundsor of the complexes containing them after administration in vitro or invivo, a cell targeting molecule or an anchoring molecule. The inventorconsequently also relates to a compound as presented above, conjugatedwith one or more targeting components via the intermediacy of at leasta) one of the carbon atoms, in particular chosen from those present onthe groups R₁, R₂ and/or R₃ or b) one of the secondary or primarynitrogen atoms of the polyamine chain. Such components may allowtargeting to a specific cell type, facilitate penetration into the cell,lysis of the endosomes or alternatively intracellular transport and arewidely described in the literature. They may be, for example, all orpart of sugars, peptides (GRP peptide, Gastrin Releasing Peptide, forexample), oligonucleotides, lipids, hormones, vitamins, antigens,antibodies, ligands specific for membrane receptors, ligands capable ofreacting with an anti-ligand, fusogenic peptides, nuclear localizationpeptides, or a combination of such compounds. There may be mentionedmore particularly the galactosyl residues which make it possible totarget the asyaloglycoprotein receptor at the surface of hepatic cells,the fusogenic peptide INF-7 derived from the influenza virushemagglutinin subunit HA-2 (Plank et al., 1994, J. Biol. Chem. 269,12918-12924) or a nuclear localization signal derived from the SV40virus T antigen (Lanford and Butel, 1984, Cell 37, 801-813) or theEpstein Barr virus EBNA-1 protein (Ambinder et al., 1991, J. Virol. 65,1466-1478).

Such conjugates can be easily obtained by techniques widely described inthe literature, and more particularly by chemical coupling, inparticular using protecting groups such as trifluoroacetyl or Fmoc orBoc, onto the polyamine. The selective deprotection of a protectinggroup then makes it possible to couple the targeting component, and theglycerolipid is then deprotected. It should be stated, however, that thesubstitution of the nonreactive groups such as the carbon atoms in theCH or CH2 groups will be carriec out during synthesis of the compoundsof the invention by methods known to a person skilled in the art whereasthe reactive groups, such as the primary or secondary amines, may be thesubject of substitutions on the neosynthesized glycerolipids of theinvention.

According to an advantageous case of the invention, said compound is ina cationic form, that is to say that it is in a form which is protonatedby binding of a proton onto one or more nitrogen atoms present on thepolyamine chain. In this case, said cationic glycerolipid is combinedwith one or more biologically acceptable anions, such as for example thetrifluoroacetate, halide, monomethylsulfate, acetate or phosphate,iodide, chloride, or bromide anion and the like. It is also possible toobtain compounds in cationic form by substitution of the amines, forexample, with a methyl or ethyl radical, and the like.

The compounds according to the present invention comprise from 2 to 7positive charges, more particularly from 3 to 5, and preferably 5. Ithas been shown that the affinity of a polyamine for DNA depends inparticular on the number of amine functional groups present on saidpolyamine (Braulin, W. H., Strick, T. J., and Record, M. T., Jr. (1982)Biopolymers 21, 1301-1314). Moreover, following their penetration intothe cell by endocytosis, the complexes formed between a DNA and acationic lipid compound are located in the endosomes in which the DNAmay be degraded under the action of pH-dependent nucleases. To counterthis phenomenon which affects the transfection efficiency, it ispossible to use lysosomotropic agents, such as for example chloroquine,whose buffering capacity at pH 5.5 makes it possible to observe animprovement in the transfection. However, the efficiency of suchcompounds is not systematic; there may be noted, by way of negativeexamples, the cases of polyethyleimine (PEI), lipospermines ordendrimers of polyamidoamine (PAMAM). Moreover, the use of chloroquineat a high dose may present a toxic risk. According to the invention, thecompounds possess a polyamine head which makes it possible to obtain asimilar effect to that of the lysosomotropic molecules. A specificadvantage of said compounds might consequently be to avoid the use ofchloroquine for the in vivo applications.

According to a preferred embodiment of the invention, said compound ischosen from the group consisting of the following formulae:

These cationic compounds may, for example, be prepared by reacting acompound of formula:

These cationic compounds may, for example, be prepared by reacting acompound of formula:

in which:

R₄ and R₅ are protecting groups forming in particular together anisopropylidene radical,

X has the same meaning as in formula I, with an acid of formula:

m and n having the same meaning as in formula I, R₆ being a protectinggroup, in particular t-butoxycarbonyl (BOC).

The functional groups O—R₄ and —O—R₅ are then deprotected so as toattach by esterification or etherification the radicals R₁ and R₂ in aknown manner.

The compound obtained is deprotected in the presence of trifluoroaceticacid. The acid of formula VII is prepared in a known manner.

In the case of the compounds of the invention for which m=3, n=2 or 3,reference will be made to the examples indicated below in order to knowthe practical modalities for the synthesis. The processes described areapplicable in general to the syntheses of the compounds according to theinvention subject to adaptations within the capability of personsskilled in the art. However, the compounds of the invention cannot belimited to those obtained by the modes of preparation described above.

According to another aspect, the invention also relates to a compositioncomprising at least one compound as described above and optionally atleast one adjuvant capable of enhancing the formation of the complexbetween a said compound and an active substance, or of enhancing thefunction of these complexes toward the cell.

Preferably, such an adjuvant will be a neutral or zwitterionic lipidwhich, for example, is or is derived from a triglyceride, a diglyceride,cholesterol (see for example U.S. Pat. No. 5,438,044), in particular, aneutral or zwitterionic lipid which is or is derived from aphosphatidylethanolamine (PE), phosphatidyl-choline, phosphocholine,sphyngomyelin, ceramide or cerebroside. Advantageously,dioleoylphosphatidylethanolamine (DOPE) will be chosen.

The weight ratio between the compound of the invention and the neutralor zwitterionic lipid is generally between 0.1 and 10, it beingunderstood that this ratio may vary depending on the nature of thecomponents considered. Persons skilled in the art have sufficientknowledge to allow these minor adaptations. It is also possible to use amixture of neutral and/or zwitterionic lipids.

The invention relates, in addition, to a complex comprising at least onecompound or at least one composition as described above and at least oneactive substance, in particular a therapeutically active substance,comprising at least one negative charge. According to a variant of theinvention, said complex may, in addition, contain one or more cationicamphiphilic agents such as those described in the literature of whichexamples were provided above. In general, there will be usedtherapeutically active substances which may be used in particular in thecontext of gene therapy.

According to a specific embodiment, said active substance is chosen fromnucleic acids and proteins. Preferably, the active substance of thecomplex according to the invention is a polynucleotide, said compound orsaid composition then making it possible to enhance the transfectingpower of the polynucleotide in a cell.

“Polynucleotide” is understood to designate a DNA and/or RNA fragmentwhich is double-stranded or single-stranded, linear or circular,natural, isolated or synthetic, designating a precise succession ofnucleotides, which are modified or otherwise (see by way of example U.S.Pat. No. 5,525,711), labeled or otherwise (see for example U.S. Pat. No.4,711,955 or EP 302175), making it possible to define a fragment or aregion of a nucleic acid without size limitation. Polynucleotide isunderstood to designate in particular a cDNA, a genomic DNA, a plasmidDNA, a messenger RNA, an antisense RNA, a ribozyme, a transfer RNA, aribosomal RNA or a DNA encoding such RNAs. “Polynucleotide” or “nucleicacid” are synonymous terms in the context of the present application.

According to a specific embodiment of the invention, said polynucleotidecomprises a gene of interest and components allowing the expression ofsaid gene of interest. In this embodiment, said polynucleotide isadvantageously in the form of a plasmid. The components allowingexpression are all the components allowing the transcription of said DNAfragment into RNA (antisense RNA or mRNA) and the translation of themRNA into a polypeptide. They are in particular promoter sequencesand/or regulatory sequences which are effective in said cell, andoptionally the sequences required to allow excretion or expression ofsaid polypeptide at the surface of the target cells. By way of example,there may be mentioned promoters such as the promoters of the virusesRSV, MPSV, SV40, CMV or 7.5 k, of the vaccinia virus, the promoters ofthe gene encoding muscle creatine kinase, actin, or pulmonarysurfactant. It is, in addition, possible to choose a promoter sequencespecific for a given cell type or which can be activated under definedconditions. The literature provides a large amount of informationrelating to such promoter sequences. Moreover, said polynucleotide maycomprise at least two sequences, which are identical or different,exhibiting a transcriptional promoter activity and/or at least twocoding DNA sequences, which are identical or different, situated,relative to each other, contiguously, far apart, in the same directionor in the opposite direction, as long as the transcriptional promoterfunction or the transcription of said sequences is not affected.Likewise, it is possible to introduce into this type of nucleic acidconstruct “neutral” nucleic sequences or introns which do not affecttranscription and are spliced before the translation step. Suchsequences and their uses are described in the literature. Saidpolynucleotide may also contain sequences required for intracellulartransport, for replication and/or for integration. Such sequences arewell known to persons skilled in the art. Moreover, the polynucleotidesaccording to the present invention may also be polynucleotides which aremodified such that it is not possible for them to become integrated intothe genome of the target cell or polynucleotides which are stabilizedwith the aid of agents such as, for example, spermine.

In the context of the present invention, the polynucleotide may behomologous or heterologous to the target cell. It may be advantageous touse a polynucleotide which encodes all or part of a polypeptide, inparticular a polypeptide having a therapeutic or prophylactic activity,and more particularly an immunogenic activity of the cellular or humoraltype. The term polypeptide is understood without restriction as to itssize or its degree of modification (for example glycosylation). Theremay be mentioned, by way of example, the genes encoding an enzyme, ahormone, a cytokine, a membrane receptor, a structural polypeptide, apolypeptide forming a membrane channel, a transport polypeptide, anadhesion molecule, a ligand, a factor for regulation of transcription,of translation, of replication, or of the stabilization of thetranscripts, or an antibody, such as for example the gene encoding theCFTR protein, dystrophin, factor VIII or IX, E6/E7 of HPV, MUC1, BRAC1,β-interferon, γ-interferon, interleukin (IL)2, IL-4, IL-6, IL-7, IL-12,tumor necrosis factor (TNF) type alpha, GM-CSF (Granulocyte MacrophageColony Stimulating Factor), the Herpes Simplex virus type 1 (HSV-1) tkgene, the gene associated with retino-blastoma or p53 or all or part ofimmunoglobulins, such as the fragments F(ab)₂, Fab′, Fab or theanti-idiotypes (U.S. Pat. No. 4,699,880). This list is of course notlimiting and other genes may be used.

According to a preferred embodiment, the complexes according to theinvention are small in size (less than 500 nm, advantageously less than200 nm and preferably less than 100 nm).

Moreover, the transfection experiments carried out show thatadvantageously the weight ratio of the lipid compound according to theinvention to said polynucleotide is 0.01 to 100. The optimum ratio isbetween 0.05 and 10.

The invention also relates to a process for preparing the complexescationic compounds/active substances comprising at least one negativecharge, said process being characterized in that one or more compoundsor compositions according to the invention are brought into contact withone or more active substances comprising at least one negative chargeand in that said complex is recovered, optionally after a purificationstep.

In a first instance, according to a first variant, one or more cationiccompounds are dissolved with an appropriate quantity of solvent ormixture of solvents which are miscible in water, in particular ethanol,dimethylsulfoxide (DMSO), or preferably a 1:1 (v:v) ethanol/DMSOmixture, so as to form lipid aggregates according to a known methoddescribed, for example, in Patent Application WO-A-9603977, or accordingto a second variant, are suspended with an appropriate quantity of asolution of detergent such as an octylglucoside such asn-octyl-β-D-glucopyranoside, or6-O-(N-heptylcarbamoyl)-methyl-α-D-glucopyranoside.

The suspension may then be mixed with a solution of active substancecomprising negative charges.

In the case where it is desirable that a neutral or zwitterionic lipidis present in the final complex, a film is formed, in the known manner,prior to the dissolution in the solvent which is miscible with water orin the solution of detergent, with a mixture containing a said cationiccompound and a said neutral or zwitterionic lipid, such as for exampleDOPE.

One of the important characteristics of the process consists in thechoice of the ratio between the positive charges of the cationic lipidand the negative charges of the active substance.

Without wishing to be limited by a specific ratio, quantities of thedifferent charges will be chosen so that the ratio between the number ofpositive charges of the cationic compound or composition and the numberof negative charges of the active substance is between 0.05 and 20, inparticular between 0.1 and 15, and preferably between 5 and 10.

The calculation to arrive at such a ratio will take into considerationthe negative charges carried by the active substance and the quantity ofcompound necessary to satisfy the ratio indicated above will beadjusted. The quantities and the concentrations for the other componentsare adjusted according to their respective molar masses and the numberof their positive and/or negative charges.

This charge ratio also constitutes an advantageous characteristic of thecomplex according to the invention.

In the case of the second variant and optionally, subsequent dialysismay be carried out in order to reduce the detergent and to recover thecomplexes. The principle of such a method is for example described byHofland et al. (1996, PNAS 93, p 7305-7309) and in chapter II of thePhilippot et al. document (G. Gregoriadis, 81-89, CRC Press 1993).

It has been shown that the first variant leads to excellent results interms of the size of the complexes obtained.

According to a third variant, one or more cationic compositions orcompounds are suspended in a buffer and then the suspension is subjectedto sonication until visual homogeneity is obtained. The lipid suspensionis then extruded through two microporous membranes under appropriatepressure. The lipid suspension is then mixed with a solution of activesubstance comprising negative charges. This so-calledsonication-extrusion technique is well known in the art.

The use of a neutral or zwitterionic lipid, such as DOPE, may proveadvantageous for the production of complexes which are small in size(less than 200 nm, preferably less than 100 nm).

The characteristics of the complexes formed may be evaluated by severalmeans which make it possible to determine, for example:

the state of complex formation with the active substance, in particularby identification of the free nucleic acids by agarose gelelectrophoresis in the case where the substances are nucleic acids,

the size of the particles by a quasi-elastic scattering of light,

the absence of precipitation over the long term.

The object of the present invention is also the complexes obtained usingthe processes listed above.

The invention also relates to the use of a compound, of a composition orof a complex according to the invention to transfer at least one activesubstance, especially a therapeutically active substance, in particulara nucleic acid, into target cells, in vitro, ex vivo or in vivo, moreparticularly in vivo.

“Target cells” according to the invention is understood to meanprokaryotic cells, yeast cells and eukaryotic cells, plant cells, humanor animal cells, and in particular mammalian cells. Cancer cells should,moreover, be mentioned. In vivo, the invention may be applied at thelevel of the interstitial or luminal space of tissues such as the lungs,trachea, skin, muscle, brain, liver, heart, spleen, bone marrow, thymus,bladder, lymph, blood, pancreas, stomach, kidney, ovaries, testicles,rectum, peripheral or central nervous system, eyes, lymphoid organs,cartilages and endothelium. According to an advantageous choice of theinvention, the target cell will be a muscle cell, a hematopoietic stemcell or alternatively a cell of the airways, more particularly atracheal or pulmonary cell, and advantageously a cell of a respiratoryepithelium.

The invention also relates to a process for transferring in vitro atherapeutically active substance into a target cell according to whichsaid cell is brought into contact with a complex according to theinvention.

The complexes according to the invention can be used as a medicament forcurative, preventive or vaccinal purposes. Accordingly, the subject ofthe invention is also the complexes of the invention as a medicament forcurative, preventive or vaccinal purposes. Such complexes may be used ina method of therapeutic treatment which consists in transferring atleast one therapeutically active substance, in particular apolynucleotide, into target cells, in particular a mammalian cell, andmore precisely a muscle cell, a hematopoietic stem cell, a cell of theairways, more particularly a tracheal or pulmonary cell, a cell of therespiratory epithelium.

More widely, the present invention also relates to a process forintroducing an active substance comprising negative charges into a cell,characterized in that cells cultured on an appropriate medium arebrought into contact with a suspension of complexes cationiccompound/active substance comprising negative charges. After a certainincubation time, the cells are washed and recovered. The introduction ofthe active substance may be checked (optionally after lysis of the cell)by any appropriate means.

The process of introduction is well known per se. The term“introduction” is understood to mean that the active substancecomprising negative charges is transferred into the cell and is located,at the end of the process, inside said cell or at the level of themembrane thereof. In the case where the active substance is a nucleicacid, reference will be made more particularly to “transfection”. Inthis case, the verification of the transfection of the nucleic acid canbe carried out by any appropriate means, for example by measuring theexpression of the gene considered or the concentration of the expressedprotein.

The invention relates more particularly to the use of a compound, of acomposition or of a complex according to the invention for thepreparation of a medicament for curative, preventive or vaccinalpurposes, intended for the treatment of the human or animal body, inparticular by gene therapy.

According to a first possibility, the medicament may be administereddirectly in vivo (for example into a muscle, into the lungs by aerosoland the like). It is also possible to adopt the ex vivo approach whichconsists in collecting cells from the patient (bone marrow stem cells,peripheral blood lymphocytes, muscle cells and the like), transfectingthem in vitro according to the present invention and readministeringthem to the patient.

The complexes according to the invention may be administered by theintramuscular, intratracheal, intranasal, intracerebral, intrapleural,intratumoral, intracardiac, intragastric, intraperitoneal, epidermal,intravenous or intraarterial route by a syringe or by any otherequivalent means, systems suitable for the treatment of the airways orof the mucous membranes such as inhalation, instillation oraerosolization. There may also be mentioned the modes of administrationby the topical route, such as for example by application of a cream, byoral administration or any other means perfectly known to the personskilled in the art and applicable to the present invention.

It is also within the scope of the invention to target specific organsor tissues by administration, in particular by the intravenous route, ofa complex according to the invention prepared so as to adjust the ratiocompound or composition/therapeutically active substance in saidcomplex, the apparent charge of the complex (see in particular Liu etal., 1997, Gene Therapy, 4, 517-523; Thierry et al., 1995, P.N.A.S., 92,9742-9746).

The invention also relates to a method of gene therapy consisting inadministering to a patient an appropriate quantity of a compositionaccording to the invention. According to the present invention and inthe context of gene therapy in vivo, it is possible to repeat severaltimes, in a given patient, the method as proposed without any majorimmune reaction being elicited against one of the compoundsadministered. The administration may take place in a single dose orrepeated once or several times after a certain time interval. Therepeated administration would make it possible to reduce the quantity oftherapeutically active substance, more particularly of DNA, to beadministered for a given dose. The appropriate route of administrationand dosage vary according to various parameters, for example theindividual or disease to be treated or alternatively the polynucleotideto be transferred.

The invention relates more particularly to a pharmaceutical preparationcomprising at least one complex as described above, optionallycontaining, in addition, at least one adjuvant capable of stabilizingsaid pharmaceutical preparation for the purpose of its storage forexample and/or of enhancing the transfecting power of said complex. Suchan adjuvant could, for example, be chosen from the group consisting ofchloroquine, a protic polar compound chosen in particular from propyleneglycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidoneor derivatives thereof, or an aprotic polar compound chosen inparticular from dimethyl sulfoxide (DMSO), diethyl sulfoxide,di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide,dimethylacetamide, tetramethylurea, acetonitrile or derivatives thereof.Likewise, said preparation may contain a pharmaceutically acceptablecarrier allowing its administration to humans or animals.

In the context of the use of a method of treatment in vivo according tothe present invention, it is, in addition, possible to carry out, beforethe administration of a pharmaceutical preparation as described above, atreatment of the patient designed to observe a temporary depletion ofthe macrophages making it possible to enhance the transfection rate.Such a technique is described in the literature; see in particular VanRooijen et al., 1997, TibTech, 15, 178-184.

The invention relates to a cell transfected with a complex as definedabove, particularly a prokaryotic cell, a yeast cell or eukaryotic cell,especially an animal cell, in particular a mammalian cell, and moreparticularly a cancer cell. According to a preferred case of theinvention, said cell is a cell of the airways, more particularly atracheal or pulmonary cell, and advantageously a cell of the respiratoryepithelium.

Finally, the invention relates to a device as well as a process allowingthe isolation of a molecule of interest containing at least one negativecharge, in particular nucleic acids as defined according to the presentinvention. “Isolation” is understood to designate the separation,detection, enrichment and purification of a fraction of anionicmolecules, according to a specific or a specific method of isolation,qualitatively and/or quantitatively.

More particularly, the invention relates to a said device consisting ofa disperse solid support, such as for example particles of polymers (ofpolystyrene, acrylamide, methacrylamide, or of one of their derivativesor any other polymer capable of forming particles of which numerousexamples are described in the literature, in particular in theliterature relating to diagnostic applications) or consisting of anondispersed solid support such as, for example, a tube, for examplemade from polystyrene, a column, for example made from hydroxyapatite, areversed phase column or an equivalent, onto which at least one compoundor one composition according to the invention is bound in its cationicform. The binding to said solid support may be achieved in a direct(adsorption for example) or indirect (via a ligand/anti-ligand typecoupling) manner. The production of such devices is within thecapability of persons skilled in the art.

Moreover, the invention relates to a process using such a device so asto allow the isolation of anionic molecules, in particular of nucleicacids. Such an isolation may in particular be nonspecific or preferablyspecific. In this second case, said compound or said cationiccomposition is, prior to the isolation step, brought into contact with,for example, an oligonucleotide whose specific sequence makes itpossible, after a hybridization step, under conditions allowing specifichybridization, to isolate in a specific manner a nucleic acid fragmentcontaining all or part of a sequence complementary to the sequence ofsaid oligonucleotide. The implementation of such a process is widelydescribed in the literature.

LEGEND TO THE FIGURES

FIG. 1: This figure illustrates the mode of synthesis of the compoundsdescribed in A.

FIGS. 2A-C: Analysis of the size of the particles of complexes formedbetween the glycerolipids of the invention and the plasmid pTG11033(Patent Application No. FR9708267) at 0.1 mg/ml which are prepared bythe ethanol technique described above. The results are presented fordifferent charge ratios. For each measurement, three independentpreparations were tested and the measurement of reproducibility isevaluated by the standard deviation between these preparations. The sizeof the particles is measured by PCS (Photon Correlation Spectroscopy).The hatched columns show the complexes obtained in the absence of DOPEand the dark columns in the presence of an equimolar quantity of DOPE.The complexes for which precipitation is observed are represented by acolumn which extends over the scale. A) glycerolipids comprising 3positive charges (C-14=pcTG 18, C-16=pcTG21, C-18=pcTG19,oleoyl=pcTG20); B) glycerolipids comprising 4 positive charges(C-14=pcTG 33, C-16=pcTG34, C-18=pcTG36, oleoyl=pcTG35); C)glycerolipids comprising oleoyl chains and 3 (pcTG20), 4 (pcTG35), or 5positive charges (pcTG56).

FIG. 3: Intravenous injection of complexes according to the invention.The luciferase activity is indicated as RLU/mg of protein).

FIG. 4: This figure illustrates the mode of synthesis of the fluorinatedglycerolipids of Example N.

FIGS. 5A to G: In vitro transfection of A549 cells in the presence ofcomplexes containing different fluorinated glycerolipids according tothe invention.

EXAMPLES

The examples below illustrate the invention, without limiting it in anymanner, with reference to the accompanying figures which form anintegral part of the description.

A. Synthesis of the Acids of Formula VII with m=3; n=2; R₆=BOC (acid 7a)or m=3; n=3; R₆=BOC (acid 7b) (see FIG. 1)

Amino acids 4, 6 and 15

A solution of acrylonitrile (9.6 ml, 146 mmol) in 50 ml of 1,4-dioxaneis added dropwise to an ice-cold solution of glycine (10.0 g, 132 mmol)and 1 N sodium hydroxide (133 ml) in a 1/1 mixture of water and1,4-dioxane (200 ml). The reaction medium is stirred at 0° C. for 1 hand at room temperature for an additional 4 h. A solution ofdi-tert-butyl dicarbonate (35.0 g, 159 mmol) in 1,4-dioxane (100 ml) isthen added drop-wise and the reaction medium is stirred for two hours atroom temperature. After extraction with ether (2×100 ml), the aqueousphase is acidified (pH 2-3) with 1 N hydrochloric acid and extractedwith ethyl acetate (2×100 ml). The combined organic phases are dried andconcentrated. The cyano acid obtained 1 (24.4 g, yield 81%) exists inthe form of a white solid of melting point 87-89° C.

¹H-NMR (200 MHz, D₂O): δ 3.88 and 3.87 (2 s, 2 H, —CH₂—CO₂H), 3.48 and3.45 (2 t, J=6.3 Hz, 2 H, —CH₂—N(BOC)—), 2.58 and 2.56 (2 t, J=6.3 and6.4 Hz, 2 H, —CH₂—CN), 1.30 and 1.24 (2s, 9 H, t-Bu-).

A solution of acid 1 (11.5 g, 50.4 mmol) in 100 ml of ethanol containing4.04 g (100 mmol) of sodium hydroxide is hydrogenated in the presence ofRaney nickel (3.2 g) for 18 h at room temperature. The mixture isfiltered on celite and the catalyst washed with methanol (2×30 ml). Thefiltrate is acidified to pH 4-5 with 10% hydrochloric acid andconcentrated under vacuum to give a white solid which is dissolved inchloroform (50 ml) in order to precipitate most of the sodium chloride.After filtration, concentration of the filtrate under vacuum andrecrystallization from carbon tetrachloride, amino acid 2 is obtained(10.4 g; yield 89%) whose melting point is 201-202° C.

¹H-NMR (200 MHz, D₂O): δ 3.53 (s, 2 H, —CH₂—CO₂H), 3.17 (t, J=6.6 Hz, 2H, —CH₂—N(BOC)—), 2.83 (t, J=7.5 Hz, 2 H, —CH₂—NH₂), 1.69 (quint, J=7Hz, 2 H, —CH₂—), 1.26 and 1.21 (2s, 9 H, t-Bu-).

The same procedure for the production of cyano acid 1 leads to theproduction of cyano acid 3 from amino acid 2.

¹H-NMR (200 MHz, CDCl₃): δ 4.00-3.85 (m, 2 H, —CH₂—CO₂H), 3.55-3.43 (m,2 H, —CH₂ —CH₂—CN), 3.31 (t, J=7.2 Hz, 4 H, —CH₂—N(BOC)—), 2.61 (m, 2 H,—CH₂—CN), 1.78 (quint, J=7.2 Hz, 2 H, —CH₂—), 1.47 and 1.44 (2s, 18 H,t-Bu-).

Amino acid 4 is obtained with a yield of 87% after purification bysilica gel chromatography (eluent:methanol/dichloromethane 3/7, then6/4) from cyano acid 3 according to the same procedure which led toamino acid 2. The melting point is 189-190° C.

¹H-NMR (200 MHz, D₂O): δ 3.57 and 3.54 (2s, 2 H, —CH₂—CO₂H), 3.2-3.0 (m,6 H, —CH₂—N(BOC)—), 2.80 (t, J=7.7 Hz, 2 H, —CH₂—NH₂), 1.80-1.50 (m, 4H, —CH₂—), 1.27 and 1.22 (2s, 18 H, t-Bu-).

The same procedure as for cyano acid 1 allows the production of cyanoacid 5 from amino acid 4.

¹H-NMR (200 MHz, CDCl₃): δ 3.85 (broad s, 2H, —CH₂—CO₂H), 3.47 (t, J=6.6Hz, 2 H, —CH₂—CH₂—CN), 3.35-3.05 (m, 8 H, —CH₂—N(BOC)—), 2.60 (m, 2 H,—CH₂—CN), 1.85-1.60 (m, 4 H, —CH₂—), 1.46 and 1.44 (2 s, 27 H, t-Bu-).

Amino acid 6 is obtained with a yield of 83% after purification bysilica gel chromatography (eluent:methanol/dichloromethane 3/7, then6/4) from cyano acid 5 according to the same procedure which led toamino acid 2.

¹H-NMR (200 MHz, D₂O): δ 3.76 and 3.73 (2s, 2 H, —CH₂—CO₂H), 3.25-2.75(m, 12 H, —CH₂—N(BOC)— and —CH₂—NH₂), 1.85-1.50 (m, 6 H, —CH₂—), 1.28and 1.23 (2s, 27 H, t-Bu-).

The same procedure as for cyano acid 1 allows the production of cyanoacid 14 from amino acid 6.

¹H-NMR (200 MHz, CDCl₃): δ 3.95 and 3.87 (2 broad s, 2 H, —CH₂ —CO₂H),3.47 (t, J=6.5 Hz, 2 H, —CH₂ —CH₂—CN), 3.40-3.05 (m, 12 H, —CH₂—N(BOC)—), 2.61 (m, 2 H, —CH₂—CN), 1.90-1.60 (m, 6 H, —CH₂—), 1.47, 1.45and 1.44 (3 s, 36 H, t-Bu-).

Amino acid 15 is obtained with a yield of 71% after purification bysilica gel chromatography (eluent:methanol/dichloromethane 1/9, then3/7) from cyano acid 14 according to a procedure identical to that whichled to amino acid 2.

¹H-NMR (200 MHz, D₂O-CD₃OD): δ 3.57 (m, 2 H, —CH ₂—CO₂H), 3.15-2.80 (m,14 H, —CH ₂—N(BOC)—), 2.70 (t, J=7.5 Hz, 2 H, —CH ₂—NH₂), 1.75-1.35 (m,8 H, —CH₂—), 1.17 and 1.13 (2 s, 36 H, t-Bu-).

Acids 7a, 7b and 7c

A solution of di-tert-butyl dicarbonate (1.09 g, 5.01 mmol) intetrahydrofuran (4 ml) is added to a solution of amino acid 4 (1.50 g,3.85 mmol) and triethylamine (1.07 ml, 7.7 mmol) in a 1/1 mixture oftetrahydrofuran and water (8 ml). The reaction medium is stirred for 2 hat room temperature. It is then acidified to pH 3 with a 10% aqueoussolution of hydrochloric acid and is extracted with ethyl acetate. Theorganic phase is washed with water, dried over sodium sulfate andconcentrated to give a colorless oil which, after chromatography on asilica gel column (eluent:ethyl acetate), gives acid 7a (1.79 g; yield:95%).

¹H-NMR (200 MHz, CD₃OD): δ 3.79 (s, 2 H, —CH₂—CO₂H), 3.30-3.15 (m, 6 H,—CH₂—N(BOC)—), 3.03 (t, J=6.7 Hz, 2 H, —CH₂—N(BOC)—), 1.85-1.60 (m, 4 H,—CH₂—), 1.46 and 1.43 (2 s, 27 H, t-Bu-).

The same procedure as for acid 7a allows the production of acid 7b (7.31g; yield: 95%) from amino acid 6 (6.50 g).

¹H-NMR (200 MHz, CDCl₃): δ 3.93 and 3.86 (m, 2 H, —CH₂—CO₂H), 3.40-3.05(m, 12 H, —CH₂—N(BOC)—), 1.85-1.60 (m, 6 H, —CH₂—), 1.44 (broad s, 36 H,t-Bu).

The same procedure as for acid 7a allows the production of acid 7c (3.37g; yield: 92% after silica gel chromatography;eluent:methanol/dichloromethane 5/95, then 10/90) from amino acid 15(3.20 g; 4.55 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 3.85 (m, 2 H, —CH₂ —CO₂H), 3.45-3.05 (m, 16H, —CH₂ —N(BOC)—), 1.85-1.60 (m, 8 H, —CH₂—), 1.45, 1.44 and 1.43 (3 s,45 H, t-Bu).

Synthesis of Cationic Glycerolipids Esters 8a, 8b and 8c

A solution of dicyclohexylcarbodiimide (0.60 g, 2.9 mmol) in drydichloromethane (1 ml) is added to a solution of acid 7a (1.10 g, 2.25mmol), (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (0.39 g, 2.9 mmol)and 4-(dimethylamino)pyridine (27 mg, 0.23 mmol) in dry dichloromethane(3 ml). The reaction mixture is stirred for 18 h at room temperature.The dicyclohexylurea precipitate is removed by filtration and thefiltrate is concentrated under vacuum and then chromatographed on asilica gel column (eluent:ether/hexane 6/4) to give ester 8a (0.72 g;53%).

¹H-NMR (200 MHz, CDCl₃): δ 4.40-4.02 (m, 4 H, —CH₂—O—), 3.98 and 3.90 (2broad s, 2 H, —CH₂—CO₂—), 3.73 (m, 1 H, CH—O—), 3.35-3.00 (m, 8 H),1.85-1.55 (m, 4 H, —CH₂—), 1.45, 1.43 and 1.42 (3 s, 30 H), 1.36 (s, 3H, Me-).

The same procedure as for ester 8a allows the production of ester 8b(3.08 g; yield: 49%) from acid 7b (5.32 g, 8.22 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 4.40-4.02 (m, 4 H, —CH₂—O—), 3.99 and 3.91 (2broad s, 2 H, —CH₂C—O₂—), 3.73 (m, 1 H, CH—O—), 3.32-3.00 (m, 12 H,—CH₂—N(BOC)—), 1.85-1.55 (m, 6 H, —CH₂—), 1.45, 1.44, 1.43 and 1.41 (4s, 39 H, t-Bu- and Me), 1.36 (2 s, 3 H, Me-).

The same procedure as for ester 8a allows the production of ester 8c(1.73 g; yield: 95%) from acid 7c (1.60 g, 1.99 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 4.35-4.04 (m, 4 H, CH₂ —O—), 3.99 and 3.92 (2s, 2 H, —CH₂—CO₂—), 3.74 (m, 1 H, >CH—O—), 3.30-3.00 (m, 16 H, —CH₂—N(BOC)—), 1.85-1.55 (m, 8 H, —CH₂—), 1.46, 1.45, 1.44 and 1.42 (4 s, 48H, t-Bu- and Me-), 1.36 (s, 3 H, Me-).

Dihydroxyesters 9a, 9b and 9c

A solution of ester 8a (663 mg, 1.10 mmol) and 1 N hydrochloric acid(0.44 ml) in methanol (19 ml) is stirred for 16 h at room temperature.Triethylamine (0.5 ml) is then added to neutrality. Evaporation undervacuum, followed by chromatography on a silica gel column (eluent:etherthen ethyl acetate) gives dihydroxyester 9a (497 mg; yield: 80%) in theform of a colorless oil.

¹H-NMR (200 MHz, CDCl₃): δ 4.26 (m, 2 H, —CH₂—OC(═O)—), 4.00-3.50 (m, 5H, CH—OH, —CH₂OH and —CH₂—CO₂—), 3.40-3.00 (m, 8 H, —CH₂—N(BOC)—),1.85-1.55 (m, 4 H, —CH₂—), 1.45 and 1.43 (2 s, 27 H, t-Bu-).

The same procedure as for dihydroxyester 9a allows the production ofdihydroxyester 9b (340 mg; yield: 71%) from ester 8b (500 mg, 0.66mmol).

¹H-NMR (200 MHz, CDCl₃): δ 4.26 (m, 2 H, —CH₂—OC(═O)—), 4.00-3.40 (m, 5H, CH—OH, —CH₂OH and —CH₂—CO₂—), 3.40-3.00 (m, 12 H, —CH₂—N(BOC)—),1.85-1.55 (m, 6 H, —CH₂—), 1.46, 1.45 and 1.43 (3 s, 36 H, t-Bu-).

The same procedure as for dihydroxyester 9a allows the production ofdihydroxyester 9c (1.23 g; yield: 83% after silica gel chromatography;eluent:methanol/dichloromethane 5/95) from ester 8c (1.55 g; 1.69 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 4.25 (m, 2 H, —CH₂—OC(═O)—), 4.00-3.40 (m, 5H, CH—OH, —CH₂ OH and —CH₂ —CO₂—), 3.40-3.00 (m, 16 H, —CH₂ —N(BOC)—),1.90-1.60 (m, 8 H, —CH₂—), 1.46, 1.45, 1.44 and 1.42 (4 s, 45 H, t-Bu-).

Triesters 10a, 11a, 12a and 13a

A solution of dicyclohexylcarbodiimide (142 mg, 0.69 mmol) in drydichloromethane (1 ml) is added to a 35 solution of dihydroxyester 9a(130 mg, 0.23 mmol), oleic acid (195 mg, 0.69 mmol; Fluka puriss.) and4-(dimethylamino)pyridine (3 mg, 0.02 mmol) in dry dichloromethane (2ml). The reaction medium is stirred for 16 h at room temperature. Thedicyclohexylurea precipitate is removed by filtration and the filtrateis concentrated under vacuum and chromatographed on a silica gel column(eluent:ether/hexane 3/7, then 4/6) to give triester 10a (142 mg; 57%)in the form of a colorless oil.

¹H-NMR (200 MHz, CDCl₃): δ 5.34 (m, 4 H, —CH═), 5.26 (m, 1 H,CH—OC(═O)—), 4.40-4.05 (m, 4 H, —CH₂—OC(═O)—), 3.95 and 3.88 (2 m, 2 H,—N(BOC)—CH₂—CO₂—), 3.35-3.00 (m, 8 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz,4 H, —CH₂—CO₂—), 2.01 (m, 8 H, —CH₂ —CH═), 1.85-1.50 (m, 8 H, —CH₂—),1.46, 1.44 and 1.42 (3 s, 27 H, t-Bu-), 1.30 and 1.27 (2 broad s, 44 H,—CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

The same procedure as for triester 10a makes it possible to obtaintriesters 11a (yield: 65%), 12a (yield: 64%) and 13a (yield: 58%) fromdihydroxyester 9a and respectively from myristic acid, palmitic acid andstearic acid.

11a ¹H-NMR (200 MHz, CDCl₃): δ 5.26 (m, 1 H, CH—OC(═O)—), 4.45-4.05 (m,4 H, —CH₂—OC(═O)—), 3.95 and 3.88 (2 m, 2 H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 8 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—),1.85-1.50 (m, 8 H, —CH₂—), 1.46, 1.44 and 1.42 (3 s, 27 H, t-Bu-), 1.26(s, 40 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

12a ¹H-NMR (200 MHz, CDCl₃): δ 5.26 (m, 1 H, CH—OC(═O)—), 4.40-4.05 (m,4 H, —CH₂—OC(═O)—), 3.95 and 3.88 (2 m, 2 H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 8 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—),1.85-1.50 (m, 8 H, —CH₂—), 1.46, 1.44 and 1.42 (3 s, 27 H, t-Bu-), 1.25(s, 48 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

13a ¹H-NMR (200 MHz, CDCl₃): δ 5.26 (m, 1 H, CH—OC(═O)—), 4.40-4.05 (m,4 H, —CH₂—OC(═O)—), 3.95 and 3.88 (2 m, 2 H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 8 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—),1.85-1.50 (m, 8 H, —CH₂—), 1.46, 1.44 and 1.42 (3 s, 27 H, t-Bu-), 1.26(s, 56 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

Triesters 10b, 11b, 12b and 13b

The same procedure as for triester 10a allows the production of triester10b (137 mg; yield: 61%) from dihydroxyester 9b (130 mg, 0.18 mmol) andoleic acid (153 mg, 0.54 mmol; Fluka puriss.).

¹H-NMR (200 MHz, CDCl₃): δ 5.34 (m, 4 H, —CH═), 5.26 (m, 1 H,CH—OC(═O)—), 4.40-4.05 (m, 4 H, —CH₂—OC(═O)—), 3.96 and 3.89 (2 m, 2 H,—N(BOC)—CH₂—CO₂—), 3.35-3.00 (m, 12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz,4 H, —CH₂—CO₂), 2.01 (m, 8 H, —CH₂ —CH═), 1.85-1.50 (m, 10 H, —CH₂—),1.46, 1.45, 1.44 and 1.41 (4 s, 36 H, t-Bu-), 1.30 and 1.27 (2 broad s,44 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

The same procedure as for triester 10a makes it possible to obtaintriesters 11b (yield: 64%), 12b (yield: 58%) and 13b (yield: 63%) fromdihydroxyester 9b and respectively from myristic acid, palmitic acid andstearic acid.

11b ¹H-NMR (200 MHz, CDCl₃): δ 5.26 (m, 1 H, CH—OC(═O)—), 4.40-4.05 (m,4 H, —CH₂—OC(═O)—), 3.97 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—),1.85-1.50 (m, 10 H, —CH₂—), 1.46, 1.45, 1.44 and 1.41 (4 s, 27 H,t-Bu-), 1.26 (s, 40 H, —CH₂—), 0.88 (t, J=25 6.4 Hz, 6 H, Me-).

12b ¹H-NMR (200 MHz, CDCl₃): δ5.26 (m, 1 H, CH—OC(═O)—), 4.40-4.05 (m, 4H, —CH₂—OC(═O)—), 3.97 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—), 3.35-3.00(m, 12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 1.85-1.50(m, 10 H, —CH₂—), 1.46, 1.45, 1.44 and 1.41 (4 s, 27 H, t-Bu-), 1.25 (s,48 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

13b ¹H-NMR (200 MHz, CDCl₃): δ 5.26 (m, 1 H, CH—OC(═O)—), 4.40-4.05 (m,4 H, —CH₂—OC(═O)—), 3.97 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—),3.35-3.00 (m, 12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—),1.85-1.50 (m, 10 H, —CH₂—), 1.46, 1.45, 1.44 and 1.41 (3 s, 27 H,t-Bu-), 1.25 (s, 56 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

Triesters 10c

The same procedure as for triester 10a allows the production of triester10c (0.75 g; yield: 47%) from dihydroxyester 9c (1.00 g; 1.14 mmol) andoleic acid (0.97 g; 3.42 mmol; Fluka puriss.).

¹H-NMR (200 MHz, CDCl₃): δ 5.34 (m, 4 H, —CH═), 5.26 (m, 1 H,CH—OC(═O)—), 4.40-4.05 (m, 4 H, —CH₂—OC(═O)—), 3.95 and 3.89 (2 m, 2 H,—N(BOC)—CH₂ —CO₂—), 3.35-3.00 (m, 16 H, —CH₂ —N(BOC)—), 2.31 (t, J=7.5Hz, 4 H, —CH₂—CO₂—), 2.01 (m, 8 H, —CH₂ —CH═), 1.85-1.50 (m, 12 H,—CH₂—), 1.46, 1.44, 1.43 and 1.41 (4 s, 45 H, t-Bu-), 1.30 and 1.27 (2broad s, 44 H, —CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

Glycerolipids pcTG20

Triester 10a (120 mg, 0.11 mmol) in solution in dry dichloromethane (1ml) is treated for 3 h with a 1/1 mixture of trifluoroacetic acid anddry dichloromethane (20 ml) at 0° C. Hexane (50 ml) is then added andthe mixture is evaporated under vacuum and gives a film which issuspended (vortex) in distilled ether (5 to 10 ml). Filtration gives awhite powder which is washed with ether and dried under vacuum to givethe glycerolipid pcTG20 (124 mg; 99%). Melting point: decomposes at 160°C.

¹H-NMR (200 MHz, CDCl₃-CF₃CO₂D): δ 5.34 (m, 5 H, —CH═ and CH—OC(═O)—),4.60-4.15 (m, 4 H, —CH₂—OC(═O)—), 3.99 (broad s, 2 H, —NH₂^(⊕)—CH₂—CO₂—), 3.45-3.20 (m, 8 H, —CH₂—NH₂ ^(⊕)—), 2.39 (t, J=7.5 Hz, 4H, —CH₂—CO₂—), 2.50-2.20 (m, 4 H, —CH₂—CH₂ ^(⊕)—NH₂ ^(⊕)—), 2.01 (m, 8H, —CH₂ —CH═), 1.61 (m, 4 H, —CH ₂—CH₂—CO₂—), 1.30 and 1.27 (2 s, 44 H,—CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-).

Elemental analysis: calculated for C₅₃H₉₂F₉N₃O₁₂: C, 56.12%; H, 8.18%;N, 3.70%. Found: C, 56.3%; H, 7.9%; N, 3.7%.

Glycerolipid pcTG35

The same procedure as for the cationic lipid pcTG20 allows theproduction of the cationic lipid pcTG35 (101 mg; yield: 97%) fromtriester 10b (100 mg, 0.08 mmol). Melting point: decomposes at 190° C.¹H-NMR (200 MHz, CDCl₃-CF₃CO₂D): δ 5.35 (m, 5 H, —CH═ and CH—OC(═O)—),4.60-4.15 (m, 4 H, —CH₂—OC(═O)—), 4.00 (broad s, 2 H, —NH₂^(⊕)—CH₂—CO₂—), 3.45-3.10 (m, 12 H, —CH₂—NH₂ ^(⊕)—), 2.40 (t, J=7.5 Hz,4 H, —CH₂—CO₂—), 2.45-2.20 (m, 6 H, —CH₂—CH₂—NH₂ ^(⊕)—), 2.01 (m, 8 H,—CH₂ —CH═), 1.60 (m, 4 H, —CH₂—CH₂—CO₂—), 1.30 and 1.27 (2 s, 44 H,—CH₂—), 0.87 (t, J=6.4 Hz, 6 H, Me-).

Elemental analysis: calculated for C₅₈H₁₀₀F₁₂N₄O₁₄: C, 53.36%; H, 7.72%;N, 4.29%. Found: C, 53.2%; H, 7.6%; N, 4.3%. The mass spectrum wasmeasured at 849.8 Da (calculated: 849.3 Da).

Glycerolipid pcTG56

The same procedure as for the glycerolipid pcTG20 allows the productionof the glycerolipid pcTG56 (0.51 g; yield: 93%) from triester 10c (0.52g; 0.37 mmol). Melting point: decomposes at 220° C.

¹H-NMR (200 MHz, CDCl₃-CF₃CO₂D) δ 5.36 (m, 5 H, —CH═ and CH—OC(═O)—),4.60-4.15 (m, 4 H, —CH₂—OC(═O)—), 4.00 (broad s, 2 H, —NH₂ ^(⊕)—CH₂—CO2—), 3.45-3.10 (m, 16 H, —CH ₂—NH₂ ^(⊕)—), 2.41 (t, J=7.5 Hz, 4 H,—CH₂—CO₂—), 2.28 (m, 8 H, —CH₂ —NH₂ ^(⊕)—), 2.01 (m, 8 H, —CH₂ —CH═),1.61 (m, 4 H, —CH₂ —CH₂—CO₂—), 1.30 and 1.27 (2 s, 44 H, —CH₂—), 0.87(t, J=6.4 Hz, 6 H, Me-).

Glycerolipids pcTG18, pcTG21 and pcTG19

The same procedure as for the glycerolipid pcTG20 makes it possible toobtain the glycerolipids pcTG18 (yield: 97%; solid; decomposes at 165°C.), pcTG21 (yield: 96%; solid; decomposes at 170° C.) and pcTG19(yield: 92%; solid; decomposes at 186° C.) from respectively triesters11a, 12a and 13a.

pcTG18 ¹H-NMR (200 MHz, CDCl₃-CF₃CO₂D ): δ 5.35 (m, 1 H, CH—OC(═O)—),4.60-4.15 (m, 4 H, —CH₂—OC(═O)—), 3.97 (broad s, 2 H, —NH₂^(⊕)—CH₂—CO₂—), 3.45-3.15 (m, 8 H, —CH₂—NH₂ ^(⊕)—), 2.38 (t, J=7.5 Hz, 4H, —CH₂—CO₂—), 2.45-2.20 (m, 4 H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.60 (m, 4 H,—CH₂ —CH₂—CO₂—), 1.25 (s, 40 H, —CH₂—), 0.87 (t, J=6.4 Hz, 6 H, Me-).

Mass spectrum: calculated 684.1 Da; measured 684.5 Da. pcTG21 ¹H-NMR(200 MHz, CDCl₃-CD₃OD ): δ 5.20 (m, 1 H, CH—OC(═O)), 4.45-4.00 (m, 4 H,—CH₂—OC(═O)—), 3.78 (broad s, 2 H, —NH₂ ^(⊕)—CH₂—CO₂—), 3.15-2.40 (m, 8H, —CH₂—NH₂ ^(⊕)—), 2.24 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.18-1.92 (m, 4H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.52 (m, 4 H, —CH₂ —CH₂—CO₂—), 1.17 (s, 48 H,—CH₂—), 0.79 (t, J=6.4 Hz, 6 H, Me-).

Mass spectrum: calculated: 740.2 Da; measured 740.6 Da. pcTG19 ¹H-NMR(200 MHz, CDCl₃-CF₃CO₂D ): δ 5.38 (m, 1 H, CH—OC(═O)—), 4.60-4.15 (m, 4H, —CH₂—OC(═O)—), 4.01 (broad s, 2 H, —NH₂ ^(⊕)—CH₂—CO₂—), 3.45-3.20 (m,8 H, —CH₂—NH₂ ^(⊕)—), 2.41 (t, J=7.5 Hz, 4H, —CH₂—CO₂—), 2.50-2.25 (m, 4H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.61 (m, 4 H, —CH₂ —CH₂—CO₂—), 1.26 (s, 56 H,—CH₂—), 0.88 (t, J=6.4 Hz, 6 H, Me-). Mass spectrum: calculated 796.3Da; measured 796.8 Da.

Glycerolipids pcTG33, pcTG34 and pcTG36

The same procedure as for the glycerolipid pcTG20 makes it possible toobtain the glycerolipids pcTG33 (yield: 97%; solid; decomposes at 205°C.), pcTG34 (yield: 94%; solid; decomposes at 215° C.) and pcTG36(yield: 92%; solid; decomposes at 220° C.) from respectively triesters11b, 12b and 13b.

pcTG33 ¹H-NMR (200 MHz, CDCl₃-CF₃CO₂D): δ 5.37 (m, 1 H, CH—OC(═O)—),4.60-4.15 (m, 4 H, —CH₂—OC(═O)—), 4.00 (broad s, 2 H, —NH₂^(⊕)—CH₂—CO₂—), 3.45-3.15 (m, 12 H, —CH₂—NH^(⊕) ₂—), 2.39 (t, J=7.5 Hz,4 H, —CH₂—CO₂—), 2.45-2.17 (m, 6 H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.60 (m, 4 H,—CH₂—CH₂—CO₂—), 1.25 (s, 40 H, —CH₂—), 0.87 (t, J=6.4 Hz, 6 H, Me-).

Mass spectrum: calculated 741.2 Da; measured 741.6 Da. pcTG34 ¹H-NMR(200 MHz, CDCl₃-CF₃CO₂D): δ 5.37 (m, 1 H, CH—OC(═O)—), 4.60-4.15 (m, 4H, —CH₂—OC(═O)—), 3.98 (broad s, 2 H, —NH₂ ^(⊕)—CH₂—CO₂—), 3.45-3.10 (m,12 H, —CH₂—NH₂ ^(⊕)—), 2.39 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.45-2.15 (m,6 H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.60 (m, 4 H, —CH₂ —CO₂—), 1.25 (s, 48 H,—CH₂—), 0.87 (t, J=6.4 Hz, 6 H, Me-).

Mass spectrum: calculated 797.3 Da; measured 797.8 Da. pcTG36 ¹H-NMR(200 MHz, CDCl₃-CF₃CO₂D): δ 5.35 (m, 1 H, CH—OC(═O)—), 4.60-4.15 (m, 4H, —CH₂—OC(═O)—), 3.98 (broad s, 2 H, —NH₂ ^(⊕)—CH₂—CO₂—), 3.45-3.10 (m,12 H, —CH₂—NH₂ ^(⊕)—), 2.37 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.45-2.15 (m,6 H, —CH₂ —CH₂—NH₂ ^(⊕)—), 1.59 (m, 4 H, —CH₂ —CH₂—CO₂—), 1.25 (s, 56 H,—CH₂—), 0.87 (t, J=6.4 Hz, 6 H, Me-). Mass spectrum: calculated 854.4Da; measured 854.0 Da.

Glycerolipid pcTG22

A slightly different route to that described above allowed theproduction of the glycerolipid pcTG22 from 3-amino-1,2-propanediol.

¹H-NMR (200 MHz, CDCl₃-CD₃OD): δ 5.04 (m, 1 H, CH—OC(═O)—), 4.27-3.92(m, 2 H, —CH₂—OC(═O)—), 3.63 (broad s, 2 H, —CH₂—C(═O)—NH—), 3.10-2.90(m, 8 H, —CH₂—NH₂ ^(⊕)—), 2.24 and 2.23 (2 t, J=7.5 Hz, 4 H, —CH₂—CO₂—),2.15-1.95 (m, 4 H, —CH₂ —CH₂—NH₂ ^(⊕)), 1.51 (m, 4 H, —CH₂ —CH₂—CO₂—),1.17 (s, 56 H, —CH₂—), 0.79 (t, J=6.7 Hz, 6 H, Me-). Mass spectrum:calculated 795.3 Da; measured 795.3 Da.

Glycerolipid pcTG90

A procedure similar to that described for the synthesis of the lipidpcTG56 allows the production of the lipid pcTG90 using3-amino-1,2-propanediol in place of glycerol.

B: Preparation of the Glycerolipid-DNA Complexes by Dissolution inEthanol

1. Preparation of the Complexes Glycerolipids pcTG20, OptionallyDOPE,-DNA

The quantities of lipids are calculated based on the concentration offinal DNA (0.1 mg/ml for the tests in cell cultures), the desired chargeratio, the molar mass and the number of positive charges of the chosencationic lipid. To obtain a complex between pcTG20/DOPE and the plasmidDNA in a ratio of 10 between positive charges provided by the cationiclipid and negative charges provided by the DNA at a final DNAconcentration of 0.1 mg/ml, the different ingredients are mixedaccording to the following calculation:

0.1 mg of DNA/ml, that is to say (0.1/330) mmol of negative charges (330Da is the average molecular weight of a nucleotide) per ml correspondto: 0.30 μmol/ml of negative charges. To obtain 10 times more positivecharges, a concentration of 3.0 μmol/ml of positive charges provided bythe cationic lipid is required. The molar mass of pcTG20 intrifluoroacetate form is 1134 g/mol and the molecule content 3 positivecharges. Therefore, 1.0 μmol/ml of pcTG20 is required, which correspondsto 1.13 mg/ml.

To obtain an equimolar concentration ofL-α-dioleoyl-phosphatidylethanolamine (DOPE, 744 g/mol, Sigma; P0510),0.74 mg/ml is required in the lipid preparation. The quantities and theconcentrations for the other compounds are adjusted according to theirrespective molar masses and the number of their positive charges.

The lipids taken up in chloroform are evaporated and then solubilized inchloroform:methanol (v:v) and again evaporated. The cationic lipids areweighed and the quantity of DOPE is added from a stock solution of 10 or20 mg/ml in chloroform to a glass tube sterilized with alcohol and withUV in order to obtain a cationic lipid concentration of 2 mM. Thesolvents are evaporated under vacuum (200 mbar) for 45 min at 45° C.using a vortex of 40 revolutions per minute (Labconco, Rapidvap,Uniequip, Martinsried, Germany). The lipid film is taken up in ethanolso as to be at the cationic lipid concentration of 50 mg/ml.

pcTG20/DOPE 1.13 mg +0.74 mg=1.87 mg in 23 μl of ethanol. This solutionis adjusted to 230 μl with 20 mM HEPES pH 7.5 in order to prepare acationic lipid solution at 5 mg/ml final.

The plasmid DNA is prepared in a plastic tube from a stock solution at 1mg/ml (10 mM Tris, 1 mM EDTA, pH 7.5).

For a solution of 0.5 ml final, 50 μl of the stock solution (50 μg DNA)are collected to which 335 μl of 20 mM HEPES pH 7.5 are added. Tocomplex the DNA with the lipid preparations, the lipids are added to theDNA. The suspension is mixed by aspiration/discharge using a pipette (10times). The complexes are stored at +4° C.

115 μl of pcTG20/DOPE are added to the 385 μl of the DNA solution inorder to obtain 0.5 ml of complex at 0.1 mg/ml DNA and at a charge ratioof 10.

The preparation of the complexes is carried out under a laminar flowcabinet.

The complexes are obtained whose characteristics are indicated in TableI below.

2. Preparation of the Complexes Glycerolipids pcT35, pcTG22 and pcTG18Optionally DOPE,-DNA

Based on the same protocol, the complexes are obtained whosecharacteristics are indicated in Table I below.

C. Preparation of the Glycerolipid-DNA Complexes by Suspension in aDetergent Solution

1. Preparation of the Complexes Glycerolipids pcTG20, OptionallyDOPE,-DNA

The quantities of lipids are calculated as described above based on theconcentration of final DNA (0.1 mg/ml for the tests in vitro), thedesired charge ratio, the molar mass and the number of positive chargesof the cationic lipid chosen. The lipids are mixed in a glass tube,sterilized with alcohol and with UV, in order to obtain a 2 mM cationiclipid solution (see above). The solvents are evaporated and the lipidfilm is taken up in a solution of n-octyl, β-D-glucopyranoside(octylglucoside, Sigma, 0 9882) according to a cationic lipid/detergentratio of 1:5 (mol:mol).

253 μl of a 20 mM octylglucoside solution in 20 mM HEPES pH 7.5 arecollected and used to take up the film of pcTG20/DOPE lipid mixture. Theplasmid DNA is prepared from a stock solution of plasmid DNA at 1 mg/mlof which 50 μl are placed in 0.5 ml (0.1 mg/ml final) to which 323.5 μlof 20 mM HEPES pH 7.5 are added. 126.5 μl of the lipid suspension areadded to the DNA by aspirating and discharging 10 times using a pipettein order to obtain the final suspension at 0.1 mg/ml of DNA and a +/−charge ratio of 10. To remove the detergent, a dialysis of 3 times 4hours at room temperature against 20 mM HEPES pH 7.5 is carried out indialysis microbags (cut-off of 13.2 kD; Sartorius, Gottingen, Germany).The dialyzed DNA/lipid complexes are stored at +4° C. The preparation iscarried out in a laminar flow cabinet.

The complexes are obtained whose characteristics are indicated in TableI below.

2. Preparation of the Complexes Glycerolipids pcTG35, OptionallyDOPE,-DNA

The same protocol is applied as above.

D. Preparation of the Lipid-DNA Complexes by Sonication Extrusion

The quantities of lipids are calculated as described above based on theconcentration of final DNA (0.1 mg/ml for the tests in vitro), thedesired charge ratio, the molar mass and the number of positive chargesof the cationic lipid chosen. The lipids are mixed in a glass tube,sterilized with alcohol and with UV, in order to obtain a 2 mM cationiclipid solution, as indicated above. The solvents are evaporated and thelipid film is taken up in 900 μl of 20 mM HEPES pH 7.5 at 4° C. forabout 16 h. The suspension is sonicated in a sonication bath (Bransonic221) to visual homogeneity.

The lipid suspension is extruded through two membranes with a porediameter of 0.2 μm (Nucleopore, Costar, Cambridge, Mass., USA) andrinsed with 20 mM HEPES pH 7.5 (extruder from Lipex Biomembranes,Vancouver, Canada) at a maximum pressure of 50 bars. The lipidsuspension is kept at room temperature for 1 hour. 450 μl of the lipidsuspension are added to 50 μl of a stock solution of plasmid DNA (1mg/ml) and mixed by aspirating/discharging 10 times using a pipette. Thelipid/DNA complexes are stored at +4° C. The preparations are carriedout under a laminar flow cabinet.

E. Protocol for Evaluation of the Complexing of the DNA by the Lipids

A 1% (w:v) agarose gel is prepared in a TAE buffer (TAE: Tris 4.8g/l+sodium acetate 0.68 g/l+EDTA 0.336 g/l pH 7.8). If necessary, thesample is diluted in TAE and then the sample buffer (0.083% bromophenolblue, 0.083% cyanol xylene FF, 10% glycerol in water) is added so as tohave 50 ngDNA/μl. The sample is briefly homogenized by vortex and leftfor 30 min at room temperature. As a control, the non-complexed plasmidprepared at the same concentration is used. 10 μl (500 ng of DNA) aredeposited on the gel and the migration is carried out at 60 mV for 3hours. The gel is developed in TAE containing 0.006% (v:v) of ethidiumbromide at 10 mg/ml for at least 30 min. Next, the gel is rinsed in TAEand analyzed under UV.

F. Protocol for Measuring the Size of the Particles by Quasi-elasticScattering of Light

The analyses are carried out on a Coulter N4Plus (Coultronics France S.A., Margency, France) at 25° C. after equilibration of the sample for 20min. An aliquot of the sample is aspirated and discharged several timesbefore being pipetted. The sample is diluted in the measuring tank andhomogenized. The measurement of the light diffracted at 90° is carriedout for 180 sec after a 180 sec wait. The range used goes from 3 nm to10,000 nm using 31 bins. To be valid, the sample should give between50,000 and 1,000,000 counts/sec.

G. Physicochemical Characteristics

The three methods of formulation “injection of ethanol”, “dialysis ofdetergent” and “sonication/extrusion” are applied to the cationic lipidsaccording to the invention with or without equimolar quantities of DOPEat charge ratios of about 10, or 5. The formulations are considered tobe appropriate when the DNA is completely complexed (no migration in theagarose gel) and when the complexes have a diameter, determined byquasi-elastic scattering of light, less than 500 nm. The tablesummarizes the results of these analyses. All the DNA/lipid complexesindicated in the table complex the DNA completely at the charge ratiosanalyzed.

TABLE I Cationic glycerolipid Ratio¹ Size (nm)² Formulation pcTG20/DOPE10 84 ± 20 ethanol pcTG20/DOPE 10 212 ± 141 detergent pcTG20/DOPE  5 96± 17 ethanol pcTG35/DOPE 10 67 ± 22 ethanol pcTG35/DOPE  5 93 ± 39ethanol pcTG35 10 83 ± 11 ethanol pcTG35  5 69³ ethanol pcTG33 10 223 ±138 sonication pcTG33/DOPE 10 243 ± 172 sonication pcTG22 10 79 ± 59ethanol pcTG22  5 87 ± 52 ethanol pcTG22/DOPE 10 261 ± 98  sonicationpcTG22/DOPE 10  89 ± 146 ethanol pcTG22/DOPE  5 72 ± 44 ethanol pcTG1810 68 ± 53 ethanol pcTG18  5 72 ± 30 ethanol pcTG18/DOPE 10 62 ± 43ethanol pcTG18/DOPE  5 109 ± 64  ethanol ¹Ratio between the positivecharges of the cationic lipid and the negative charges of the DNA.²Determined 24 to 48 hours after the preparation (± represents thestandard deviation of the measurement). ³Determined 8 days after thepreparation

These analyses show that the formulations meet the necessaryrequirements. The “injection of ethanol” method gives the best resultsfor numerous preparations which are less than 100 nm in size. Themethods by dialysis of detergent and sonication/detergent also make itpossible to obtain complexes meeting the objectives of the presentinvention.

H. In Vitro Transfection of Satellite Cells

Cultures of dog muscle and human muscle cells are carried out in an HamF14 medium (Life Technologies) supplemented with 10% fetal calf serum(FCS, Hyclone, Logan, Utah), 10 μg/ml of insulin (Sigma), 10 ng/ml ofEGF (Sigma) and of FGF (Pepro Tech Inc, Rocky Hill, N.J.) 2 mM ofglutamine (bioMérieux), and 40 μg/ml of gentamycin (Schering Plough).

The cells are inoculated 24 h to 48 h before the transfection into a96-well culture plate with about 5×10³ to 10⁴ cells per well, at about30% confluence, and kept at 37° C. under a 5% CO₂ and 95% airatmosphere.

The transfections are carried out with mixtures of variable quantitiesof lipids and plasmid DNA in order to determine the charge ratios andthe optimum DNA concentrations per well.

The complexes used are prepared 24 h to 48 h before the transfection anddiluted in HamF 14 plus 40 μg/ml of gentamycin and 2 mM glutamine.

After removing the culture medium, 100 μl of transfection mixtures withor without 10% FCS are transferred into each of the wells and the platesare incubated for 4 h at 37° C.

All the transfection media are then adjusted to 10% FCS, 10 μg/ml ofinsulin (Sigma), 10 ng/ml of EGF (Sigma) and of FGF (Pepro Tech Inc,Rocky Hill, N.J.), 2 mM glutamine (bioMérieux), and 40 μg/ml ofgentamycin (Schering Plough) for a final volume of 250 μl. The culturesare incubated for 48 h and then the cells are recovered and tested fortheir capacity to express the luciferase gene. The proteinconcentrations are determined by the system for testing quantity ofprotein (Promega).

I. Transfection of A549 Cells with Lipid Complexes

The A549 cells (epithelial cells derived from human pulmonary carcinoma)are cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10%fetal calf serum (Gibco BRL) 24 hours before the start of thetransfection in 96-well plates (2×10⁴ cells per well) in a humidatmosphere at 37° C. and 5% CO₂/95% air. For the transfection in theabsence of serum, the medium is removed and replaced with serum-freemedium. In another microplate, the following suspensions of lipid/DNAcomplexes are prepared (lipid/DNA complexes at 0.1 mg/ml of DNA and atthe indicated charge ratio): 44 μl (4.4 μg DNA), 22 μl (2.2 μg DNA), 5.5μl (0.55 μg DNA) of stock solution in the first 3 wells, and 11 μl (0.11μg DNA) of the stock solution diluted 10-fold in the next well. Thevolume is adjusted to 110 μl with DMEM and 100 μl are transferred overthe A549 cells. The incubation is carried out with 4, 2, 0.5 and 0.1 μgof DNA per well for 4 hours. Next, 50 μl of DMEM+30% fetal calf serumare added 4 hours after the start of transfection and then 100 μl ofDMEM+10% FCS 24 hours after the start of transfection. The transfectionsin the presence of 10% fetal calf serum are carried out in an identicalmanner except that the transfection occurs in medium with serum.

J. Analysis of the Transfection

48 h after the transfection, the medium is removed and the cells arewashed with 100 μl of PBS phosphate solution and lyzed with 50 μl oflysis buffer (Promega). The lysates are frozen at −80° C. until theexpressed luciferase activity is measured. The latter is carried out on20 μl of mixture for one minute using the “Luciferase” determination kit(Promega) (LB96P Berthold luminometer) in 96-well plates in kineticmode.

K. Transfection in Vitro

A few of these preparations were evaluated in transfection in vitrousing the A549 cells and the dog primary satellite cells.

The results are summarized in Table II below and show the relative lightunits (RLU) per well. The values given are obtained with 2 μg of DNA perwell. All the complexes are prepared using the injection of ethanolmethod. The total protein concentration per well is determined by theconventional techniques (BCA test, Pierce). As a guide, a well containsabout 20 to 30 μg of protein.

TABLE II A549 + myoblasts Lipid Ratio¹ A549² serum myoblasts + serumpcTG20/DOPE 10  3.6 × 10⁵ 6.1 × 10⁶ 8.9 × 10⁶ 2.3 × 10⁷ pcTG20/DOPE  58.25 × 10⁶ 2.8 × 10⁷ 8.8 × 10⁵ 1.3 × 10⁶ pcTG35/DOPE 10   4 × 10⁶ 1.0 ×10⁷ 7.1 × 10³ 6.5 × 10⁷ pcTG35/DOPE  5  3.3 × 10⁷ 6.8 × 10⁷ 4.3 × 10⁶1.0 × 10⁸ pcTG35 10  1.2 × 10⁸ 7.6 × 10⁶ 1.8 × 10⁷ 6.2 × 10⁸ pcTG35  5 1.6 × 10³ 6.6 × 10⁷ 3.4 × 10⁸ 1.7 × 10⁸ ¹Ratio between the positivecharges of the cationic lipid and the negative charges of the DNA. ²FreeDNA between 0 and 270 relative light units.

The expression of luciferase (RLU/min) reported in mg of proteins givesthe following values:

pcTG35 R+/−10 (A549) 3.4×10¹⁰ RLU/min/mg protein

pcTG35 R+/−5 (A549) 2.8×10¹⁰ RLU/min/mg protein

pcTG35/DOPEv R+/−5 (A549+serum) 1.0×10¹⁰ RLU/min/mg protein

The compounds pcTG20 and pcTG35 are capable of efficiently transfectingthe two types of cells tested when they are complexed with the plasmidDNA. It will be noted that some formulations give higher results in thepresence of serum and that the serum has no inhibitory effect on thistype of preparation.

In the same manner, analyses of transfection in vitro which were carriedout on A549 cells have made it possible to demonstrate a) that thecompound pcTG90 complexed with a plasmid pTG11033 makes it possible toobserve an efficient transfection under similar conditions to thosedescribed above in the absence or in the presence of DOPE, in theabsence or in the presence of serum and b) to verify that DOPE could besubstituted by another adjuvant such as for example1,2-di-stearoyl-sn-glycerophosphoethanolamine (Avanti 850715),1,2-di-phytanoyl-sn-glycero-3-phosphoethanolamine (Avanti 850402),1,2-di-myristoyl-sn-glycero-3-phosphoethanolamine (Avanti 850745),1,2-di-lauroyl-sn-glycero-3-phosphoethanolamine (Avanti 850702),1,2-di-palmitoyl-sn-glycero-3-phosphoethanolamine (Avanti 850705),1,2-di-elaidoyl-sn-glycero-3-phosphoethanolamine (Avanti 850725),1,2-di-palmitoleyl-sn-glycero-3-phosphoethanolamine (Avanti 850706) or1,2-di-linoleoyl-sn-glycero-3-phosphoethanolamine (Avanti 850755).

L. Analysis of the Size of the Particles (according to the protocoldescribed in F).

The analyses carried out by PCS show that the size of the particles andtheir aggregation strongly depend on the charge ratio, the structure ofthe lipid as well as the presence or absence of DOPE.

Stable complexes of 100 to 200 nm were reproducibly obtained for acharge ratio of 10 when the cationic glycerolipid is in excess relativeto the DNA (see FIGS. 2). The results show that only the complexescontaining a C18 glycerolipid (pcTG19, pcTG36) have a problem ofsuspension, giving rise to large complexes whose size is variable. Incontrast, the oleoyl-type lipids containing 4 or 5 charges (pcTG35,pcTG56) make it possible to obtain complexes of about 200 nm for acharge ratio of 5. Moreover, we showed that an equimolar quantity ofDOPE slightly increases the size of the particles and reduces thetendency which the complexes have to aggregate at a low charge ratio(see FIGS. 2A and 2B). DOPE makes it possible, in addition, to obtain astabilizing effect for the complexes containing C-14 or C-16glycerolipids with three amine groups (FIG. 2A); this effect is notobserved for the homologous compounds containing four amine functionalgroups (FIG. 2B). The tendency of the complexes to aggregateconsiderably increases for a charge ratio of 5, suggesting that therepulsions of charges is a decisive factor for avoiding the aggregationphenomenon. Comparison of the complexes containing glycerolipidscarrying fatty acids of increasing length show minor differences betweenthe C-14, C-16 and oleoyl derivatives for a charge ratio of 10, inparticular in the case of the glycerolipids containing 4 amine groups.The number of amine groups at the level of the polar head of theglycerolipids containing oleoyls exhibits a slight effect on the size ofthe complexes (FIG. 2C).

It should be noted, in addition, that the results observed by thistechnique for measuring the size of the complexes were confirmed byelectron microscopy.

M. Intravenous Injection of Complexes According to the Invention

The results are summarized in FIG. 3. Complexes according to theinvention were synthesized according to the methods described above fromthe glycerolipids pcTG35, pcTG56 and pcTG90, in the presence of anequimolar quantity of DOPE, at a fixed charge ratio of 5, using aplasmid containing the gene for luciferase pTG11033 (French PatentApplication No. 97/08267).

The mice used are 9 to 11-week old female C57BL/6 mice. The intravenousinjections are performed in the tail after disinfecting the skin with70% ethanol. The volume injected is 200 μl and the DNA concentration is0.24 mg/ml (lipid concentration 10 mg/ml).

Two days after the injections, the mice are sacrificed. Afterextraction, the tissues are frozen in liquid nitrogen and stored at −80°C. In order to measure the luciferase activity, the tissues aremechanically ground with the aid of a pestle in a mortar placed on dryice. 500 μl or 200 μl of lysis buffer (Promega) are added to the tissuedebris obtained from lungs or trachea, respectively, and subjected tothree freeze/thaw stages. The cellular debris is removed bycentrifugation and the luciferase activity (in RLU/min, relative lightunit per minute) is measured on 20 μl of supernatant in accordance withthe manufacturer's instructions (Promega) by adding 100 μl of reagentand by measuring the activity by luminescence. The luciferase activitymeasured is standardized relative to the protein quantity with the aidof a calibration series prepared from commercially available luciferase(Promega). The total protein quantity is, moreover, determined by thecolorimetric bicinchoninic acid BCA method (Smith et al., 1985, Anal.Biochem., 150, 76-85 Pierce) using one aliquot of supernatant. Thismakes it possible to express the luciferase activity in RLU permilligram of protein extracted from the tissues.

The results show that the expression of the luciferase reporter gene inthe lungs after intravenous injection of complexes containing one of thethree glycerolipids indicated above in the presence of DOPE is markedlyenhanced relative to the injection of DNA alone by a factor of about 15to 30 times. The values indicated are mean values obtained from 2 to 6mice injected.

N Synthesis of Fluorinated Glycerolipids pcTG69 to pcTG72.

1. Synthesis of the Triesters (see FIG. 4)

Triester 6: 232 mg (1.12 mmol) of dicyclohexylcarbodiimide in 1 ml ofdichloromethane are added to a solution of dihydroxyester 5 (270 mg,0.37 mmol), fluorinated acid 1 (454 mg, 1.12 mmol) and4-(dimethylamino)pyridine (5 mg, 0.038 mmol) in 4 ml of dichloromethane.The reaction is carried out with stirring for 16 hours at roomtemperature. The dicyclohexylurea precipitate is removed by filtrationand the filtrate is concentrated under vacuum and subjected tochromatography on a silica gel column (eluent:ether:hexane, 4:6, v:v) soas to obtain triester 6 (275 mg, 48%) in the form of a viscous liquid.¹H-NMR (200 MHz, CDCl₃): δ 5.25 (m, 1 H, >CH—OC(O)—), 4.40-4.08 (m, 4 H,—CH₂—OC(O)—), 3.95 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—), 3.35-3.00 (m,12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.20-1.88 (m, 4H, —CH₂—CF₂—), 1.82-1.50 (m, 10 H, —CH₂—), 1.45, 1.44, 1.43 and 1.41 (4s, 36 H, t-Bu-), 1.29 (br s, 28 H, —CH₂—), ¹⁹F NMR (376 MHz, CDCl₃): d−81.6, −115.1, −125.0, −126.5.

Triester 7: According to an identical protocol to that described fortriester 6, triester 7 (220 mg, 49%) is obtained from dihydroxyester 5(170 mg, 0.236 mmol) and fluorinated acid 2 (426 mg, 0.707 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 6.40 (dtt, J=15.6, 6.9, 2.2 Hz, 2 H,═CH—CF₂—), 5.59 (dt, J=15.6, 12.3 Hz, 2 H, ═CH—CH₂—), 5.25 (m, 1H, >CH—OC(O)—), 4.40-4.05 (m, 4 H, —CH₂—OC(O)—), 3.96 and 3.89 (2 m, 2H, —N(BOC)—CH₂—CO₂—), 3.35-3.00 (m, 12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.2Hz, 4 H, —CH₂—CO₂—), 2.20 (m, 4 H, allylic H), 1.80-1.50 (m, 10 H,—CH₂—), 1.45, 1.44, 1.43 and 1.41 (4 s, 36 H, t-Bu-), 1.30 (br s, 20 H,—CH₂—). ¹⁹F NMR (376 MHz, CDCl₃): d −81.3, −111.6, −121.9, −122.4,−123.2, −123.9, −126.6.

Triester 8: According to an identical protocol to that described fortriester 6, triester 8 (230 mg, 47%) is obtained from dihydroxyester 5(210 mg, 0.291 mmol) and fluorinated acid 3 (441 mg, 0.874 mmol).

¹H-NMR (200 MHz, CDCl₃) δ 5.25 (m, 1 H, >CH—OC(O)—), 4.40-4.10 (m, 4 H,—CH₂—OC(O)—), 3.95 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—), 3.35-3.00 (m,12 H, —CH₂—N(BOC)—), 2.31 (t, J=7.5 Hz, 4 H, —CH₂—CO₂—), 2.20-1.90 (m, 4H, —CH₂—CF₂—), 1.82-1.50 (m, 10 H, —CH₂—), 1.45, 1.44, 1.43 and 1.41 (4s, 36 H, t-Bu-), 1.29 (br s, 28 H, —CH₂—). ¹⁹F NMR (376 MHz, CDCl₃): d−81.3, −114.9, −122.4, −123.2, −124.0, −126.6.

Triester 9: According to an identical protocol to that described fortriester 6, triester 9 (165 mg, 49%) is obtained from dihydroxyester 5(160 mg, 0.222 mmol) and fluorinated acid 4 (280 mg, 0.666 mmol).

¹H-NMR (200 MHz, CDCl₃): δ 5.25 (m, 1 H, >CH—OC(O)—), 4.45-4.08 (m, 4 H,—CH₂—OC(O)—), 3.95 and 3.89 (2 m, 2 H, —N(BOC)—CH₂—CO₂—), 3.35-3.00 (m,12 H, —CH₂—N(BOC)—), 2.37 (m, 4 H, —CH₂—CO₂—), 2.26-1.95 (m, 4 H,—CH₂—CF₂—), 1.85-1.55 (m, 14 H, —CH₂—), 1.45, 1.44, 1.43 and 1.41 (4 s,36 H, t-Bu-). ¹⁹F NMR (376 MHz, CDCl₃) d −81.3, −114.9, −122.4, −123.4,−124.1, −126.7.

2. Synthesis of pcTG69

Triester 6 (120 mg, 0.079 mmol) in 1 ml of dichloromethane is treatedfor 3 hours with 16 ml of a solution of trifluoroacetic acid anddichloromethane (1:1, v:v) at 0° C. 100 ml of hexane are added and themixture is evaporated under vacuum so as to obtain a film which is takenup in distilled ether. After filtration, a white powder is obtainedwhich is washed with ether and dried under vacuum to give the compoundpcTG69 (115 mg, 94%). Mp=205° C.

3. Synthesis of pcTG70

The glycerolipid pcTG70 (185 mg, 86%) is obtained in the form of a whitepowder from triester 7 (210 mg, 0.111 mmol) according to a processidentical to that described for pcTG69. Mp=210° C.

4. Synthesis of pcTG71

The glycerolipid pcTG71 (141 mg, 90%) is obtained in the form of a whitepowder from triester 8 (150 mg, 0.089 mmol) according to a processidentical to that described for pcTG69. Mp=220° C.

5. Synthesis of pcTG72

The glycerolipid pcTG72 (87 mg, 92%) is obtained in the form of a whitepowder from triester 9 (90 mg, 0.06 mmol) according to a processidentical to that described for pcTG69. Mp=220° C.

These fluorilated compounds correspond to the backbone of pcTG35 (4amino groups) with a polycarbon chain of variable size (C11, C15, C17 orC19 unsaturated). They were synthesized according to the injection ofethanol method.

O. In Vitro Transfection of the Complexes Containing the FluorilatedCompounds

The efficacy of the complexes formed according to the preceding methoddescribed with the fluorinated compounds (see Example N) was studied ona culture of A549 cells according to the technique described in ExampleI. The charge ratios tested are chosen among the ratios 10, 5, 2.5, 1.25and 0.8 (see FIGS. 5A to G). The tests were also carried out in theabsence or in the presence (indicated in the figures by ser.) of serumor of DOPE, and for various quantities of DNA (0.1, 0.5, 2 or 4 μg). Theresults (FIGS. 5 A to G) show that:

pcTG69 (FIG. 5C) makes it possible to observe, under the conditionstested, luciferase expression levels comparable to those obtained withnonfluorinated analogous C14 or C16 compounds. Moreover, other results,not shown in FIG. 5, showed that the best results were obtained withpcTG69 with a charge ratio of 2.5, in the presence or in the absence ofDOPE or of serum,

pcTG72 (FIGS. 5A and B) makes it possible to obtain satisfactorytransfection rates for a charge ratio of 10, in the presence or in theabsence of DOPE or of serum; on the other hand, for a charge ratio of1.25 or 0.8, it is observed that the presence of DOPE is a factor whichmakes it possible to enhance the transfection rate, in particular in thepresence of quantities of DNA greater than 0.1 μg,

pcTG71 (FIGS. 5D and E) allow suitable transfection rates to be obtainedonly in the presence of DOPE, for charge ratios of 1.25, 0.8 or 10, inparticular for quantities of DNA greater than 0.5 μg. Under theconditions tested, transfections in the presence of DOPE do not appearto be affected by the presence of serum in the medium,

pcTG70 (FIGS. 5F and G): For charge ratios of 10 or 5, the transfectionrates observed are effective, in the presence or in the absence of DOPEor of serum. On the other hand, for lower charge ratios (1.25 or 0.8),the presence of DOPE appears to be required.

What is claimed is:
 1. A complex comprising (i) at least one compound offormula I:

in which: R₁ and R₂, which are identical or different, are C₆-C₂₃ alkylor alkenyl radicals which are linear or branched, or radicals—C(═O)-(C₆-C₂₃) alkyl or —C(═O)-(C₆-C₂₃) alkenyl which are linear orbranched, X is an oxygen atom or an amino radical —NR₃, R₃ being ahydrogen atom or an alkyl radical having 1 to 4 carbon atoms, n is apositive integer from 1 to 6, m is a positive integer from 1 to 6, andwhen n>1, m may be identical or different, wherein said compound is in acationic form, and (ii) at least one active substance comprising atleast one negative charge.
 2. The complex of claim 1, wherein R₁ and R₂,which are identical or different, are linear —C(═O) alkyl or linear—C(═O) alkenyl radicals.
 3. The complex of claim 1, wherein R₁ and R₂,which are identical or different, are —C(═O) alkyl or —C(═O) alkenylradicals comprising from 12 to 20 carbon atoms.
 4. The complex of claim1, wherein n is an integer chosen from the numbers 2, 3 or
 4. 5. Thecomplex of claim 1, wherein m is an integer chosen from the numbers 2, 3or
 4. 6. The complex of claim 1, wherein R₁, R₂ or a combination thereofis fluorinated.
 7. The complex of claim 6, wherein the number offluorinated carbon atoms on R₁, R₂ or a combination thereof may rangefrom 1 to
 12. 8. The complex of claim 6, wherein R₁, R₂ or a combinationthereof are alkyl radicals having 15 carbons and the number offluorinated carbon atoms on the chains R₁, R₂ or a combination thereofis
 4. 9. The complex of claim 1, wherein the N—H and NH₂ groups aresubstituted with methyl or ethyl radicals instead of hydrogen.
 10. Thecomplex of claim 1, wherein said compound comprises from 2 to 7 positivecharges.
 11. The complex of claim 1, wherein said compound has a formulaselected from the group consisting of:


12. The complex of claim 1, further comprising at least one adjuvantwhich enhances the formation of the complex between said compound andsaid active substance.
 13. The complex of claim 12, wherein saidadjuvant is a neutral or zwitterionic lipid.
 14. The complex of claim13, wherein said neutral or zwitterionic lipid is or is derived from atriglyceride, a diglyceride, cholesterol, a phosphatidylethanolamine(PE), phosphatidylcholine, phosphocholine, sphygomyelin, ceramide orcerebroside.
 15. The complex of claim 14, wherein said neutral orzwitterionic lipid is dioleylphosphatidylethanolamine (DOPE).
 16. Thecomplex of claim 14, wherein the compound/adjuvant weight ratio isbetween 0.1 and
 10. 17. The complex of claim 1, wherein said activesubstance is a nucleic acid comprising a cDNA, a genomic DNA, a plasmidDNA, an antisense polynucleotide, a messenger RNA, a ribosomal RNA, aribozyme, a transfer RNA, or a DNA encoding such RNAs.
 18. The complexof claim 1, wherein said complex has a size of less than 500 nm.
 19. Thecomplex of claim 1, wherein said complex has a size of less than 100 nm.20. The complex of claim 1, wherein a ratio between positive charges ofsaid cationic compound(s) and negative charges of said active substanceranges from 0.05 to
 20. 21. A composition comprising an effective amountof the complex of claim 1 and a pharmaceutically acceptable carriertherefor.
 22. The composition of claim 21, further comprising at leastone adjuvant which enhances the transfective power of said complex. 23.The composition of claim 21, wherein said adjuvant compriseschloroquine, a protic polar compound selected from the group consistingof propylene glycol, polyethylene glycol, glycerol, ethanol,1-methyl-L-2-pyrrolidone and a derivative thereof, or an aprotic polarcompound selected from the group consisting of dimethyl sulfoxide(DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone,sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea,acetonitrile and a derivative thereof.
 24. A method of deliverycomprising transferring at least one therapeutically active substanceinto target cells along with an effective amount of the compound ofclaim
 1. 25. The method of claim 24, wherein said target cell is amammalian cell.
 26. The method of claim 25, wherein said target cell isselected from a muscle cell, a hematopoietic stem cell, or a cell of theairways.
 27. The method of claim 26, wherein said cell of the airways isselected from a tracheal or pulmonary cell.
 28. A method of delivery oftherapeutically active substances in the human or animal body, saidmethod comprising administering the complex of claim 1 to a human oranimal in need of such prevention or treatment.
 29. The method of claim27, wherein said complex is administered by intramuscular injection, byinhalation, by intratracheal injection, by instillation, byaerosolization, by the topical route or by the oral route.
 30. Thecomplex of claim 1, wherein said compound is conjugated with one or moretargeting components via at least one carbon atom from those present ingroups R₁, R₂, or R₃.
 31. The complex of claim 1, wherein said compoundis conjugated with one or more targeting components via at least onesecondary or primary nitrogen atom.